**5. Conclusions**

*Leishmania* parasites express proteins belonging to the SUMO protein modification pathway. The gene coding for SUMO is essential for growth and/or viability of *L. donovani* promastigotes, while the SENP processing enzyme is required for the C-terminal processing of SUMO and its nuclear localization, but dispensable for *L. donovani* viability. The SENP−/<sup>−</sup> null mutants show a 60% reduced growth at ambient temperature, but less impact at mammalian tissue temperature. No decrease of viability during *in vitro* infection can be observed, indicating a primary role for SENP-dependent SUMOylation in the fast growing promastigote stage. Additionally, the viability of SENP−/<sup>−</sup> null mutants hints at a vital importance of as yet unknown, SENP-independent functions of SUMO.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2073-4425/11/10/1198/s1, Table S1: Oligonucleotides used for targeting constructs; Table S2. Primers used for RT-qPCR and PCR; and Figure S1: Images of original Western blot and Coomassie Brilliant Blue-stained PA gel.

**Author Contributions:** A.B.: gene replacements, transgene expression and phenotype analyses, imaging, draft manuscript. C.K .-B.: gene , experimental design, imaging, draft manuscript, supervision. C.B.: NGS analysis, growth kinetics. M.S.: gene replacements. J.C.: study design, supervision, artwork, manuscript conception and finalization. All authors have read and agreed to the published version of the manuscript.

**Funding:** A.B. is funded by the Joachim Herz Graduate School of Infection Biology at the Bernhard Nocht Institute for Tropical Medicine, Hamburg. No further external funding was received.

**Acknowledgments:** We thank D. Çadar for the use of the Illumina MiSeq system.

**Conflicts of Interest:** The authors declare no conflict of interest.
